coloc2 plugin

[Laure Twyffels]

Dear all,

 

I have a few questions about the output of the coloc2 plugin.

 

1)      How are the “Ch1 Max threshold” and “Ch2 Max threshold” calculated?
Do they correspond to Costes thresholding?  

2)      How are the Pearson’s “below threshold” and “above threshold” R
values calculated? Do they correspond to the values obtained for the pixels
in which at least one channel is below its “Max threshold” and for the
pixels in which both channels are above their “Max thresholds”,
respectively? Which use would you make of these values?

3)      Is Costes thresholding used for the Manders “threshold”
coefficients?

 

And finally, a more general question: I have analyzed only a few images
until now, but for all of them, and all my ROIs, I get a message saying that
“The absolute y-intercept of the auto threshold regression line is too
high”, and suggesting that I should use a ROI or do a background
subtraction. However, the values of the y-intercept do not seem to me
excessively high (e.g. -1, +3); what I consider as background has a low mean
value too, even inside ROIs. Also, I feel it strange to subtract a
background before asking the plugin to calculate a threshold that is used to
determine what “background” is. What is your opinion about that?

 

Thank you for your help !

 

Laure

 

Laure Twyffels

Center for Microscopy and Molecular Imaging (CMMI)

Université Libre de Bruxelles

 


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[Ellen Arena-2]

First off - sorry for the delay in answering…

To hopefully help answer your questions Laure… see below for my attempts to answer your specific questions.  I believe there are others on this listserv who are in better command of Coloc2 (I am only just recently getting into the code myself) and colocalization in general.

To start with some helpful links - but hopefully, you’ve found these already:
http://imagej.net/Coloc_2 <http://imagej.net/Coloc_2>
http://imagej.net/Colocalization_Analysis <http://imagej.net/Colocalization_Analysis>

Also - the ImageJ Forum has been getting some recent activity in regards to colocalization analyses…  It might be a good option for posting questions as well - I know some of the Coloc2 folks are currently active on there.  So might be worth posting these questions there as well to get some more helpful answers.
http://forum.imagej.net/ <http://forum.imagej.net/>  

Ellen

> On Jan 21, 2016, at 4:33 AM, Laure Twyffels <[hidden email]> wrote:
>
> Dear all,
>
>
>
> I have a few questions about the output of the coloc2 plugin.
>
>
>
> 1)      How are the “Ch1 Max threshold” and “Ch2 Max threshold” calculated?
> Do they correspond to Costes thresholding?  

The quick answer to this one is ‘yes’ - they are relying on the regression line… and within the code it is specified that:
"A class implementing the automatic finding of a threshold used for Pearson colocalisation calculation."
After looking through the code a bit - I believe those thresholds are calculated via the Costes method.

>
> 2)      How are the Pearson’s “below threshold” and “above threshold” R
> values calculated? Do they correspond to the values obtained for the pixels
> in which at least one channel is below its “Max threshold” and for the
> pixels in which both channels are above their “Max thresholds”,
> respectively? Which use would you make of these values?
>

Honestly… I am not as familiar with the code for the Pearson’s calculations… from what I can tell - they are checking whether or not the calculated thresholds in either channel is above/below 0.  but honestly - I would have to defer to the Coloc2 experts to best answer this question.  Sorry!


> 3)      Is Costes thresholding used for the Manders “threshold”
> coefficients?

Yes.  From Costes paper - these values are Thresholded split Manders' colocalization coefficients.

>
>
>
> And finally, a more general question: I have analyzed only a few images
> until now, but for all of them, and all my ROIs, I get a message saying that
> “The absolute y-intercept of the auto threshold regression line is too
> high”, and suggesting that I should use a ROI or do a background
> subtraction. However, the values of the y-intercept do not seem to me
> excessively high (e.g. -1, +3); what I consider as background has a low mean
> value too, even inside ROIs. Also, I feel it strange to subtract a
> background before asking the plugin to calculate a threshold that is used to
> determine what “background” is. What is your opinion about that?
>

From what I understand… even ‘low’ background levels can greatly affect the calculations for coloc parameters.  MCC are particularly sensitive to non-zero background - as background is assumed ‘0’ for these calculations. So in a way - the background subtraction that you carry out will determine the validity of MCC as a measure of colocalization.  I know - this is not so straightforward an answer… and reveals the sensitivity in these coloc measurements - especially at the time of acquisition if the offset is not set properly, etc.  I would say - subtracting background is a necessary step in general - especially if you have this non-zero background.  As long as you do so in a reproducible manner - and document the steps you carry out - it is fine.  The links to the imagej wiki I gave you above should also reveal some of these issues with background and interpretation of coloc values.

Again - perhaps others with more experience/expertise can chime in here…  (or try the forum post!!!).

>
>
> Thank you for your help !
>
>
>
> Laure
>
>
>
> Laure Twyffels
>
> Center for Microscopy and Molecular Imaging (CMMI)
>
> Université Libre de Bruxelles
>
>
>
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html

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